inhalation nanomedicines – opportunities and challenges · inhalation nanomedicines –...

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Helmholtz Institute for Pharmaceutical Research Saarland (HIPS)Department of Drug Delivery (DDEL)

[email protected]

Inhalation Nanomedicines – Opportunities and Challenges

“Les traitments nebulisees en Pneumologie”Geneve, 13. January 2010

Prof. Dr. Claus-Michael Lehr

WhereWhere isis thethe Saarland Saarland locatedlocated??

2

““Drug Delivery ResearchDrug Delivery Research””

HIPS Dept. of Drug Delivery (DDEL)

Central Research Theme:„Biological Barriers“

Cell & tissue based in-vitro models– intestinal mucosa– skin– lung

Drug delivery technologies=> Explore the potential of

(nano-sized) carriers to OVERCOME thesebarriers

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Inhalation Inhalation NanopharmaceuticalsNanopharmaceuticals ––WhatWhat areare potential potential advantagesadvantages??

First First ThoughtThought::

ImprovedImproved lunglung depositiondeposition of of nanosizednanosized aerosolsaerosols (10(10--20nm) 20nm) comparedcompared to to „„classicalclassical““ pharmaceuticalpharmaceutical aerosolsaerosols (2(2--55µµm)m)

BUT: BUT: -- MakingMaking drug/carrierdrug/carrier particlesparticles thatthat smallsmall in in notnot easyeasy……-- Still Still needneed to to deliverdeliver a a significantsignificant dosedose……

meansmeans ((tootoo) ) manymany particlesparticles!!

=> => ProbablyProbably of of limitedlimited potentialpotential

Inhalation Inhalation NanopharmaceuticalsNanopharmaceuticals ––WhatWhat areare potential potential advantagesadvantages??

Second Second ThoughtThought::Controlling Controlling thethe dispositiondisposition of of drugs/particlesdrugs/particles AFTER AFTER

depositiondeposition::

–– ImproveImprove ABSORPTION ABSORPTION acrossacross thethe „„airair--bloodblood--barrierbarrier““ ((e.ge.g. to . to deliverdeliver macromolecularmacromolecular biopharmaceuticalsbiopharmaceuticals))

–– Control/avoidControl/avoid pulmonarypulmonary CLEARANCE (CLEARANCE (e.ge.g. to . to createcreate a a platformplatform forfor inhalableinhalable controledcontroled releaserelease systemssystems))

–– CellCell--specificspecific TARGETING ( TARGETING ( e.ge.g. . forfor vaccinesvaccines, , genegene therapytherapy, , antianti--cancercancer drugsdrugs))

= > = > appearsappears as as thethe majormajor potentialpotential

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OutlineOutline

Deposition of (nano) particles on mucosal epithelial Deposition of (nano) particles on mucosal epithelial cells and their effects on drug absorption cells and their effects on drug absorption

Pulmonary clearance of (NanoPulmonary clearance of (Nano--)particles )particles –– mucociliary clearancemucociliary clearance–– macrophage clearancemacrophage clearance

Cellular delivery of telomerase inhibitors by Cellular delivery of telomerase inhibitors by polymeric nanocarriers for the treatment of lung polymeric nanocarriers for the treatment of lung cancer cancer

Epithelia - The place of landing

Calu-3 (human cancercell line)Foster et al. 2000 Int J PharmFlorea et al. 2003 J Control Rel

16HBE14o- (humanimmortalised cell line)Ehrhardt et al. 2002 Cell Tissue ResEhrhardt et al. 2003 Pharm Res

HAEpC (human cellsin primary culture)Elbert et al. 1999 Pharm ResFuchs et al. 2003 Cell Tissue Res

Examples for availablein vitro models:

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TheThe alveolar alveolar epitheliumepithelium::CellularCellular morphologymorphology of a of a singlesingle alveolusalveolus

J.S. Patton, Advanced Drug Delivery Reviews 19 (1996) 3-36.

Days of culture

day0 day2 day4 day8

mea

n Fl

-1

0

5

10

15

20

25

30

proSp-Cisotypic control

Expression of Cav-1 and Sp-C in time course (FACS)

Fuchs et al., Cell & Tissue Res., 311 (2003), 31-45

Days of culture

day0 day2 day4 day8

mea

n of

Fl-1

0

50

100

150

200

250

300

cav-1isotypic control

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Evidence for nanometric vesicles in human alveolar epithelial cells in primary culture

Fuchs et al., Cell & Tissue Res. 311 (2003), 31-45

Numerous <50nm-pores make the alveolar epithelial cell membrane look like a sponge!

apical membranestight junctions

NEEDED: Barrier Properties!

A functional epithelium is more than just an assembly of cells!

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Human lung epithelial cell culture systems -Development of tight junctions:

Visualization of tight junctional protein ZO-1 by specific antibody,nuclei counterstained by propidium iodide, observed by CLSM

A549 cell line, day 8human alveolar epithelial cellsin primary culture, day 8

Elbert et al, Pharm. Res. 16 (5) (1999) 601-608.

Apical to basolateralabsorption of

Salbutamol (SAL)FITC dextran 4k (FD4)Fluorescein-Na (FLU)Atenolol (ATE)Rhodamine 123 (RHO)Budesonide (BUD)Triamcinolon-

acetonide (TAA)Propranolol (PRO)

across 16HBE14o-bronchial epithelial celllayers

Active vs. Passive Transport

Actively transported substances are outside the CI 95%

SAL

RHO

P app[cm

/sec]

log P

-3 -2 -1 0 1 2 3 41,00e-7

1,00e-6

1,00e-5

Passive Diffusionr2

= 0,9529Confidence Interval 95%Active Transport

FLU

ATE

TAA

BUDPRO

FD4

Ehrhardt et al, 2003

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What needs to be considered What needs to be considered when when studingstuding the interaction of the interaction of aerosolized (aerosolized (nano)PARTICLESnano)PARTICLES

-- rather than SOLUTES rather than SOLUTES ––

with pulmonary epithelial barriers ?with pulmonary epithelial barriers ?

Liquid interface culture

apical

basolateral

Cellmonolayer

Air interface culture

Significant consequences for cellular differentiation and proliferaftion!

KNOWN: KNOWN: EffectEffect of of CellCell CultureCulture conditionsconditions::Liquid Liquid versusversus Air Air interfaceinterface cultureculture

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Liquid interface deposition Air interface deposition

Significant consequences for transport kinetics!

NOT SO MUCH KNOWN: NOT SO MUCH KNOWN: EffectsEffects of of depositiondeposition conditionsconditions::Liquid Liquid versusversus Air Air interfaceinterface depositiondeposition

water uptake from bulk fluid water uptake from thin fluid layer covering lung epithelial cells

Absorption of water from humidified environment

Particle‐Water Interactions

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Time [hours]

0 1 2 3 4

trans

porte

d sa

lbut

amol

sul

phat

e [%

]

0

10

20

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60

70

0µl

Time [hours]

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trans

porte

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lbut

amol

sul

phat

e [%

]

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3,4µl0µl

Time [hours]

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porte

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lbut

amol

sul

phat

e [%

]

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3,4µl

6,8 µl

0µl

Time [hours]

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porte

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amol

sul

phat

e [%

]

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3,4µl

12,5 µl

6,8 µl

0µl

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trans

porte

d sa

lbut

amol

sul

phat

e [%

]

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3,4µl

12,5 µl

6,8 µl

25 µl

0µl

Time [hours]

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trans

porte

d sa

lbut

amol

sul

phat

e [%

]

0

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3,4µl

12,5 µl

6,8 µl

25 µl50 µl

0µl

Time [hours]

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trans

porte

d sa

lbut

amol

sul

phat

e [%

]

0

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3,4µl

12,5 µl

6,8 µl

25 µl50 µl100 µl

0µl

Transport of Transport of salbutamolsalbutamol sulfatesulfate (Easyhaler(Easyhaler®®) across hAEpC ) across hAEpC cell monolayers; (data present mean cell monolayers; (data present mean ±± standard deviation; n = 4)standard deviation; n = 4)

TheThe volumevolume of of thethe liquid donor liquid donor phasephase influencesinfluencesdrugdrug transporttransport acrossacross AIC AIC cellcell monolayersmonolayers

Concentrationgradient controls absorption rate!

How to reproducibly deposit aerosol particleson cell monolayers?

PennCentury DP4 Insufflator

Aerosol deposition withoutsize classification

Aerosol deposition with sizeclassification

Cell compatibleMSLI

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InfluenceInfluence of of particleparticle sizesize -- budesonidebudesonide

Transport of budesonide (Δ Cyclocaps®; • Autoinhaler®; ♦ Easyhaler® in all cases applied 30 µM) across air interface cultivated hAEpC.

Time [hours]

0 1 2 3 4tra

nspo

rted

bude

soni

de [%

]0

10

20

30

40

50

60

70

4

Time [hours]

0 1 2 3 40

10

20

30

40

50

60

70

trans

porte

d bu

deso

nide

[%]

Time [hours]

0 1 2 3 40

10

20

30

40

50

60

70

trans

porte

d bu

deso

nide

[%]

Cyclocapsx50= 55,64µm

500 µm

Easyhaler x50= 49,56µm500 µm

Autoinhalerx50= 18,00µm

500 µm

Small soluble particles yield higher absorption ratesBur et al., in press

InfluenceInfluence of of particleparticle sizesize -- salbutamolsalbutamol

Transport of salbutamol sulphate (Δ Cyclocaps®; • Ventilastin® Novolizer®; ♦ Easyhaler®, in all cases 1000 µM donor concentration) across air interface cultivated hAEpC monolayers.

Time [hours]

0 1 2 3 4

trans

porte

d sa

lbut

amol

sul

phat

e [%

]

0

1

2

3

4

5

6

7

Cyclocapsx50= 45,15µm

500 µm

Easyhalerx50= 58,33µm

500 µm

Novolizerx50= 217,58µm

500 µm

Time [hours]

0 1 2 3 40

1

2

3

4

5

6

7

trans

porte

d sa

lbut

amol

sul

phat

e [%

]

Time [hours]

0 1 2 3 40,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

trans

porte

d sa

lbut

amol

sul

phat

e [%

]

large soluble particles yields lower absorption ratesBur et al., in press

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““A new A new PPharmaceutical harmaceutical AAerosol erosol DDeposition eposition DDevice evice oon n CCell ell CCultures (PADDOCC) ultures (PADDOCC)

as alternative method for biocompatibility and as alternative method for biocompatibility and ADME screeningADME screening””

in collaboration with in collaboration with

Experimental setup

sedimentation chamberAKITA system

aerosolisationchamber

Chamber forHandiHaler

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Main components

Aerosol generation Aerosol deposition

HandiHaler

aerosol cloud

Principle of sedimentation chamber

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sedimentation chamber

Snapwell with Calu-3cell monolayer at AIC

Akita System

aerosolisation chamber

chamber with HandiHaler

Particle aerosolisation phase

chamber with HandiHaler

aerosolisation chamber

sedimentation chamber

Snapwell with Calu-3cell monolayer at AIC

Akita System

Particle sedimentation phase

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ReproducibleReproducible depositiondepositionsamesame drugdrug -- different different concentrationsconcentrations

depo

site

d am

ount

in µ

g

0

1

2

3

4

5

6

200µg 400µg 800µgbudesonide (n=11)

First First transporttransport experimentsexperiments

tim e (m in)0 50 100 150 200 250 300

trans

porte

d am

ount

in %

0

20

40

60

80

budesonide (n=12)sa lbutam ol (n=9)

air interface deposition in PADDOCC and subsequent air interface transport

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OutlineOutline


Pulmonary clearance of (NanoPulmonary clearance of (Nano--)Particles )Particles –– mucociliary clearancemucociliary clearance–– macrophage clearancemacrophage clearance


„Nanotechnology based formulations forsustained-release pulmonary drug delivery“

NanoInhale

J.V. Liebig Univ. Giessen

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Mechanisms of particle clearance from the lungs

Airways:

Particles cleared byMucociliary Clearance

Peripheral lung:

particles clearedby macrophages

Mechanisms of particle clearance from the lungs

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IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

particles :

tracheaalveoli

cilia

ciliated cells

mucus

Goal: Removal of particulate matter from the airways by coordinated transport of mucus

1. metachronal: propagation of the wave in t and x

2. 200-300 cilia per ciliated cell (Reinhardt, 2001)

3. beating-frequence 10-20 Hz (Iravani et al., 1975)

4. complex beating cycle

Mucociliary Clearance

5 Andi Henning, Julian Kirch

Controlled breeding Trachea isolation

2-3 mm

3-4 mm

Particle deposition

Oblong cut

18-22 mm

ECT – Embryonic Chicken Trachea

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Ludin chambertemperature (33°C) & humidity

(99%) controllable

particle tracking videos

transmission light & fluorescence light

9 Seminar AG Lehr 05.01.2010 Julian Kirch

Ex-vivo experiments Embryonic Chicken Trachea

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Clearance Velocity

Carbon particles, ~ 5-10µm

Is the particle size influencing clearance rate ?

Polystyrene particles / FluoresBrite™

→ Fluorescently labeled (YG - ex/em 441/486)

→ Size: 6 µm / 1 µm / 0.1 µm / 0.05 µm

No significant influence of particle size

for polystyrene particles!

0,01,02,03,04,05,06,0

0,055 0,1 1,0 6,0

Particle size [µm]

Cle

aran

ce [m

m/m

in]

21

0.0

2.0

4.0

6.0

8.0

X

Chito-X

PVA-X

DEAPA-PVA-X

P(VS-V

A)-X

PEG-10-X

PEG-5-X

Tra

nspo

rt [m

m/m

in]

N >100 Partikel

Signifcant differences in Clearance rates, butNOT due to differences in size or shape!

Effect of Polymer-Material

→ Particlesize: 83 nm < d < 389 nm

→ Zeta-Potential: -42 mV < ζ < 47 mV

0.0

2.0

4.0

6.0

8.0

X

Chito-X

PVA-X

DEAPA-PVA-X

P(VS-V

A)-X

PEG-10-X

PEG-5-X

Tra

nspo

rt [m

m/m

in]

N >100 Partikel

1) Chito-X → MC ↓↓

2) DEAPA → MC ↓

3) VS → MC ↓

4) PEG-5kDa → MC ↑↑

Effect of Polymer-Material

5 kDa 10 kDa

22

Sanderson et al., 1981

Epithelium and Mucus

two layers:

„gel“-layer: viskoelastic, non-newtonian gel, usually „mucus“

and

„sol“-layer (Periciliary Layer, PCL): watery, newtonian liquid


Surfactantlayer

mucus

nanoparticles with different vclearance ifapplied alone

interpretation of results:

hypothesis 1 („passive“, inert particles)

flow-velocity

10

Mucociliary Clearance Embryonic Chicken Trachea

Seminar AG Lehr 05.01.2010 Julian Kirch

=slowly cleared

=quickly cleared

23

11


mucus

flow-velocity



=slowly cleared

=quickly cleared


hypothesis 2 („active“/ „reactive“ particles)

11


mucus

flow-velocity



=slowly cleared

=quickly cleared


hypothesis 2 („active“/ „reactive“ particles)

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Discrimination:

„multi-tracking“: direct comparison by parallel

tracking of different particles on one trachea

advantage:

interindividual and batch-dependent variability can be

avoided

disadvantage:

no difference in clearance visible if all particles avoid

clearance (more or less) „actively“ by altering

mucus/clearance properites

rheological measurements!


Ex-vivo experiments Embryonic Chicken Trachea

„activ“ / „reactive“

„passive“ , inert



Influence of particle shape on vclearance

Spheres:

Ø 0.5 µm Polystyrene,

labelled with fluorescend dye

Rods:

made of streched Ø 0.5 µm

Polystyrene spheres

particles donated by Mitragotri-group, UCSB, Santa Barbara, USA

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spheres vs. rods(both polystyrene, labelled)

0

0,5

1

1,5

2

2,5

3

3,5

4

1

clea

ranc

e ve

loci

ty [m

m/m

in]

spheres

rods

370 particles on 5 trachea

270 particles on 5 trachea

influence of particle shape on vclearance : single tracking

spheres vs. rods MULTITRACKING(both polystyrene, labelled)

0

0,5

1

1,5

2

2,5

3

3,5

4

1

clea

ranc

e ve

loci

ty [m

m/m

in]

spheresrods



influence of particle shape on vclearance : multitracking

approx. 300 particles

on 8 trachea

approx. 300 particles

on 8 trachea

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Peripheral lung:

particles clearedby macrophages

Macrophage Clearances of Nanopharmaceuticals

Species differences relevant to alveolar clearance

Macrophages from Non-Smoker and Smoker

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The ideal model for alveolar clearance (?): „Human alveolar macrophages in primary co-culture

with autologus epithelial cell monolayers“cd68ßfitc.001

100 101 102 103 104FL1-H

M1M2

M3

FACS analysis of isolated CD 68 marked macrophages.

Coculture of human alveolar epithelial cells and macrophages.

Coculture of human alveolar epithelial cells and and human alveolar macrophages from the same individuum. Red: RCA stained epithelial cells, green: CD68 FITC stained macrophages.

0

20

40

60

80

100

120

140

160

180

200

Aden

o-Ca

Aden

o-Ca

Aden

o-Ca

Aden

o-Ca

bron

chial

alve

olar c

arcino

m

bron

chial

alve

olar c

arcino

m

Diffuse

pare

nchy

male lu

ng di

seas

e

small

cell l

ung c

ance

r

small

cell l

ung c

ance

rPE

CPE

CPE

CPE

CPE

CPE

C

IL-8

[pg/

g tis

sue]

High variability of the „quality“

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Immortalized mouse alveolar macrophages (MHS) as model for alveolar clearance

Single culture Coculture with human alveolar epithelial cells

Alveolar Clearance of Polystyrene particles10 µm1,75 µm0,1 µm

37°C

37°C +Cytochalasin

4°C

0,1µm Polystyrene particlescross the cell membran also

without active uptake.

1,75µm Particles arephagocytosed actively

(37°C).

10µm Particles circumventalveolar macrophages

clearance

Uptake of particles by MHS cells andthe mechanisms involved appear to be size dependent!

Red: RCA stained macrophages

Green: Polystyren particles

But quantitative Interpretation ???

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Macrophage particle interactions –size dependency -

MHS only 0,1µm 0,2µm 1µm 6µm

73,12% cellsare interacting

0% cellsare

interacting

100% cellsare interacting

100% cellsare interacting

AirAir

Erythrocyt Erythrocyt

Capillary endothelium

Plasma in capillary

Interstitium

AT II cell

AT I cell

The Air - Blood - Barrier

basemen

t mem

brane

- Not only an epithelial barrier... -

Phospholipid-rich interfacial filmAir

Alveolar lining fluid: 90-140 nm *~ 90% lipids (mainly phospholipids)~ 10% proteins * Green et al. The Role of Surfactant in Disease Associated

with Particle Exposure. Lung Biology in Health and Disease (2000) vol. 143 pp. 533-576

66

30

zz

AT II

AT I

AT I

Plasma

Air

„What happens after landing?“

Particle deposition

Role of Surfactant Proteins ?

SP adsorption on particles?

scale

Role of Surfactant Proteins

• Protein Corona Theory(Dawson et al.)

Dynamic layer of proteins adsorbed to nanoparticle surfaces immediately upon contact with living systems

Primary surface which is in contact with cells *

Which proteins adsorb on particles in dependence on particle material?

• Identify and quantify adsorbed surfactant proteins (especially SP-A)

• Investigate binding affinities and binding forces

How do adsorbed SP influence the biological response in terms of

• Clearance by alveolar macrophages

• Epithelial translocation

• Open questions after alveolar deposition of particles: ??

* Lynch. Protein-nanoparticle interactions. Nano Today (2008) vol. 3 (1-2) pp. 40-47

SP-A

68

31

Methodological approach

Protein component

Surfactant protein containing incubation media

Extraction of particulate matter

??

Idea: incubation of model particles with a model alveolar lining fluid

• Material

• Size & shape

• Zeta potential

• Hydrophobicity

• bBALF

Particlecomponent

Magnetic core

Polymer matrix(Pharmaceutically relevant polymers)

100 nm

69

Used magnetic nanoparticles

Chitosan Phospholipid

StarchPMO

32

SP-A adsorption on 200 nm magnetic nanoparticles

Data shown as mean ± S.E.M. (n=3)71

Particle properties determine SP-A adsorption

Surface modification Zeta-Potential [mV] Hydrophobicity [mL/µm2] SP-A [%](related to control)

Chitosan 44,0 ± 2,4 8*10-10 69,9

Phospholipid -40,9 ± 5,3 6*10-11 82,17

Poly-Maleic-Oleic Acid -21,4 ±,02 1*10-13 66,81

Oleic Acid 3*10-12

Starch -1,1 ±0,4 7*10-13 24,2

Chitosan Phospholipid PMO Starch

⊕⊖Hydrophobic interactions Electrostatic interactions

72

33

SP-A adsorption relative to nanoparticle surface (I)

~630 kD

~ 30 nm

~ 20 nm

Haagsman und Diemel. Comp BiochemPhysiol, Part A Mol Integr Physiol (2001)

vol. 129 (1) pp. 91-108

=> A ≃ 706 nm2=>d=30nm

=> Theoretical max. number of SP-A molecules per particle (monolayer): ~178 SP-A molecules / particle

Surface area per particle (d=200 nm) ≃ 0.1256 µm2

1.2 mg/mL particles (d=200 nm) ≃ 2.29*1011 particle / mL

=> Total surface area of particles Atot ≃ 28.8*109 µm2/mL

=> Theoretical mass concentration / mL of SP-A for complete coverage of all particles:

0.0426 mg SP-A73

SP-A adsorption relative to nanoparticle surface (II)

=> Theoretical mass concentration / mL of SP-A for complete coverage of all particles:

0.0426 mg SP-A

Surface modification

Absolute bound amount of SP-A [mg/mL] „Coverage factor“

Chitosan 0.186±0.033 4,36

Phospholipid 0.232±0.069 5,45

Poly-Maleic-Oleic Acid 0.193±0.066 4,53

Starch 0.118±0.019 2,77

=> Multiple layers of SP-A on each particle for

NP-to-protein ratio of 2:1 ??

74

34

Summary & Outlook• Determination of adsorbed SP-A via densitometry is possible

(at least on a semiquantitative level)

• There are differences in amount of adsorbed SP-A among the tested particles

• Protein adsorption is strongly dependent on particle material (e.g. Starch vs. Chitosan)

or ?

Future tasks:

•Adsorptionisothermes (preliminary experiments performed)

•Role of Ca2+ on SP-A adsorption („mode of binding“)

•Influence of adsorbed SP-A on clearance by alveolar macrophages (MH-S)75

OutlineOutline


Pulmonary clearance of (NanoPulmonary clearance of (Nano--)Particles )Particles –– mucociliary clearancemucociliary clearance–– macrophage clearancemacrophage clearance


35

Cationic chitosan/PLGA nanoparticles enhance the uptake of the antisense

telomerase inhibitor 2’-O-Methyl-RNA into A549 lung cancer cells

Sebastian Taetz1, Noha Nafee1, Christiane Baldes1,

Kamilla Piotrowska2, Julia Beisner2, Thomas Mürdter2,

Ulrich F. Schaefer1, Ulrich Klotz2, Claus-Michael Lehr1

1Biopharmaceutics and Pharmaceutical TechnologySaarland University, Saarbrücken

2Institute for Clinical Pharmacology, Dr. Margarete Fischer-Bosch Hospital, Stuttgart

Funding: DEUTSCHE KREBSHILFE

Why telomerase inhibition for cancer treatment?

Rest of chromosome

Telomere (TTAGGG)n

„Hayflick limit“

shortening of telomeres during cell cycles

cellular senescence

apoptosis

normal cells

36

Telomere (TTAGGG)nRest of chromosome

„Hayflick limit“

cellular senescence

apoptosis

cells expressing telomerasepermanently

TelomerasehTERT

hTR

template region

85% cancer cells are telomerase positive!

cancer cells

The inhibitorantisense 2’-O-Methyl-RNA (2OMR)

• 13mer antisense oligonucleotide

• directed against the template region of hTR

• highly selective and effective

Disadvantage:

– big molecule

– high negative charge

no uptake, i.e. hard to deliver!

Piotrowska et al. 2005, Lab InvestPitts et al. 1998, Proc Natl Acad Sci USA

37

Nanosphere-DNAComplexes

Nanospheres< 200 nm

Nanospheres< 200 nm

Surface Morphology

Chitosan coated PLGA Nanospheres

Kumar et al, Biomaterials 25 (2004) 1771-1777

Cationic chitosan/PLGA nanoparticlesfor the delivery of antisense 2‘-O-methyl-RNA

Taetz, S. et al., 2008, Eur. J. Pharm. Biopharm., in press

-----------

--

13mer2‘-O-methyl-RNA

Mr = 4430 Da

hTERT

hTR

38

Uptake of nanoplexes into A549 cells

red = DiI-stained chitosan/PLGA nanoparticles (3CPNP)green = FAM-2OMRpurple = cell nuclei

1 day after incubationoverlay FAM-2OMR DiI-CPNP

Uptake of 2OMR is mediated via nanoplexes!

Uptake of 2OMR nanoplexesinto different cells types

A549

A549

Calu-3

Calu-3

hAEpC

hAEpC

50 µm 50 µm 50 µm

50 µm 50 µm 50 µm

red = cell membrane green = FAM-2OMR

afte

rinc

ubat

ion

afte

r1 d

ay

39

Influence of 2´-O-Methyl-RNA –nanoplexeson telomerase activity and telomere length

23.1[kb]

9.4

6.6

4.3

5 10 15[weeks]

Controls

5 10 15[weeks]

Inhibitor

5 10 15

[weeks]

2

3

4

5

6

7

[kb]

p=0.036 p=0.025

p=0.0028

Beisner et al, Lung Cancer 2009Tätz et al, EJPB 2008

Tumor cells were implantedinto the flanks of nude mice.

Tumor size is measured witha calliper every 3 days

The resulting tumors weregrown (max tumor size 2 cm) H&E stained section 100x magnification

Institut für Klinisch-Experimentelle ChirurgieProf. Dr. Michael D. MengerDr. Matthias W. Laschke

Institut für Biopharmazie und Pharmazeutische. Technologie.

Prof. Dr. Claus-Michael LehrDr. Brigitta Loretz

40

Time, days

Are

a, m

m²

⇒injection of 1x106 cells results in continuos tumor growth in nude mice.

Institut für Klinisch-Experimentelle ChirurgieProf. Dr. Michael D. MengerDr. Matthias W. Laschke

Institut für Biopharmazie und Pharmazeutische. Technologie.

Prof. Dr. Claus-Michael LehrDr. Brigitta Loretz

Towards animal experiments:Growth curve NCI-H460 (=HTB-177) human lung carcinoma (large cell lung cancer)

Acknowledgements

MediTRANSTargetedDelivery of Nanomedicine

MediTRANSTargetedDelivery of Nanomedicine

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